Biological defense strategies have evolved to protect organisms from invasion by other species. Microbial infection response systems include oxidative and non-oxidative mechanisms, utilizing compounds that are enzymatically synthesized in cells and peptides that are single gene products.
Anti-microbial peptides constitute an oxygen-independent host defense system found in organisms encompassing many taxonomic families. One major class of anti-microbial peptides can be sequence-defined by conserved cysteine residue patterns and are termed defensins. Mammalian defensins, derived from skin, lung and intestine, exhibit antibiotic activity against a wide variety of pathogens, including gram-positive and gram-negative bacteria, fungi (e.g. Candida species) and viruses. See, for example, Porter et al., Infect. Immun. 65(6): 2396-401, 1997.
The amphipathic character of the defensin peptides appears to be the key to the general mechanism of microbial attack, i.e., by creating pores, or xe2x80x9cboringxe2x80x9d through the cell wall. In addition, Daher et al., J. Virol. 60(3): 1068-74, 1986, reported that enveloped viruses, including herpes simplex types 1 and 2, cytomegalovirus and influenza virus (A/WSN), among others, were inactivated by incubation with human neutrophil peptide (HNP-1) and speculated that the binding of defensin molecules to viruses impairs the virus"" ability to infect cells.
The defensin family of anti-microbial peptides can be divided into two major subclasses based on two distinct consensus sequences. See, for example, Martin et al., Journal of Leukocyte Biology 58: 128-36, 1995. The first defensin subclass, classic defensins, represented by HNPs are stored in the so-called large azurophil granules of neutrophils and macrophages and attack microorganisms that have been phagocytosed by these cells. The amino acid sequence of HNPs is, consistent with a predicted disulfide bridging that is distinct from that of the xcex2-defensin subclass. Epithelial cells can also be a source of defensins, and these cells appear to secrete these peptides into the external, extra-cellular environment. In the mouse, for example, Paneth cells of the small intestine and proximal colon, secrete defensin-like peptides, called cryptidins, into the lumen. See, for example, Ouellette and Selsted, The FASEB Journal 10: 1280-9, 1996.
xcex2-defensins, the second major defensin subclass, include peptides found in bovine lung (e.g., BNBD-bovine neutrophil xcex2-defensins) as well as a secreted form (TAP-tracheal anti-microbial peptide). See, for example, Selsted et al., J. Biol. Chem. 268(9): 6641-8, 1993. Two human xcex2-defensins have been reported. SAP-1 was isolated from human psoriatic skin, and hBD-1 was found in low concentrations in human blood filtrate. See, for example, Bensch et al., FEBS Lett. 368(2): 331-5, 1995). The amino acid sequence of these human xcex2-defensins is most similar to the bovine BNDPs and TAP. See, for example, Harder et al., Nature 387: 861, 1997, wherein SAP-1 is designated hBD-2.
Other than the conserved cysteine residues the defensin family is quite sequence divergent. It is possible that the variant amino acid positions may be related to the site or conditions of activity or to the spectrum of pathogens attacked by a particular defensin.
In addition to anti-microbial activities, particular defensins exhibit metabolically sensitive cytotoxic activity (Lichtenstein et al., Blood 68: 1407-10, 1986 and Sheu et al., Antimicrob. Agents Chemother. 28: 626-9, 1993), alter the response of adrenal cortical cells to ACTH (Zhu et al., Proc. Natl. Acad. Sci. (USA) 85(2): 592-6, 1988) and have specific chemotactic activity for human monocytes (Territo et al., J. Clin. Invest. 84(6): 2017-20, 1989). Recruitment of monocytes by neutrophils may, in part, be mediated by neutrophilic defensins and suggests a pro-inflammatory activity for these peptides in addition to their anti-microbial effects. Also, a decrease in defensin mRNA level has been demonstrated in SPG (specific granule disease). See, for example, Tamura et al., Japan. Int. J. Hematol. 59(2): 137-42, 1994. Higazi et al., J. Biol. Chem. 271(3): 17650-5, 1996, suggested that plasminogen bound to fibrin in the presence of defensin may be less susceptible to activation by tPA.
Moieties having anti-microbial, immunostimulatory, pro-inflammatory and other properties of defensins are sought. The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.
Within one aspect the invention provides an isolated protein comprising a polypeptide that is at least 80% identical to a polypeptide selected from the group consisting of: a) a polypeptide having the sequence of amino acid residue 1 to amino acid residue 65 of SEQ ID NO:2; b) a polypeptide having the sequence of amino acid residue 19 to amino acid the sequence of amino acid residue 1 to amino acid residue 67 of SEQ ID NO:10; e) a polypeptide having the sequence of amino acid residue 21 to amino acid residue 67 of SEQ ID NO:10; and f) a polypeptide having the sequence of amino acid residue 23 to amino acid residue 67 of SEQ ID NO:10; wherein the polypeptide has cysteine residues corresponding to amino acid residues 33, 40, 45, 55, 62 and 63 of SEQ ID NOs:2 or 10. Within one embodiment the protein comprises a polypeptide having the sequence selected from the group consisting of: a) a polypeptide having the sequence of amino acid residue 1 to amino acid residue 67 of SEQ ID NO:10; b) a polypeptide having the sequence of amino acid residue 21 to amino acid residue 67 of SEQ ID NO:10; and c) a polypeptide having the sequence of amino acid residue 23 to amino acid residue 67 of SEQ ID NO:10.
Within another aspect is provided an isolated protein having the sequence of SEQ ID NO:10 from amino acid residue 23 to amino acid residue 67.
Within another aspect the invention provides a polypeptide selected from the group consisting of: a) amino acid residue 30 to amino acid residue 63 of SEQ ID NO:2; b) amino acid residue 31 to amino acid residue 63 of SEQ ID NO:2; c) amino acid residue 30 to amino acid residue 64 of SEQ ID NO:2; d) amino acid residue 31 to amino acid residue 64 of SEQ ID NO:2; and e) a polypeptide chosen from SEQ ID NOs:14-72.
Within still another aspect is provided a pharmaceutical composition comprising a polypeptide selected from the group consisting of: a) a protein according to claim 1, b) amino acid residue 30 to amino acid residue 63 of SEQ ID NO:2; c) amino acid residue 31 to amino acid residue 63 of SEQ ID NO:2; d) amino acid residue 30 to amino acid residue 64 of SEQ ID NO:2; e) amino acid residue 31 to amino acid residue 64 of SEQ ID NO:2; and f) a polypeptide chosen from SEQ ID NOs:14-72; in combination with a pharmaceutically acceptable vehicle.
Within yet another aspect is provided an antibody that specifically binds to a protein as described above.
Within a further aspect is provided an anti-idiotypic antibody of an antibody which specifically binds to a protein as described above.
Within another aspect is provided an isolated polynucleotide molecule encoding a protein, the polynucleotide molecule consisting of a coding strand and a complementary non-coding strand, wherein the polynucleotide molecule encodes a is polypeptide that is at least 80% identical to the amino acid sequence to a polypeptide selected from the group consisting of: a) a polypeptide having the sequence of amino acid residue 1 to amino acid residue 65 of SEQ ID NO:2; b) a polypeptide having the sequence of amino acid residue 19 to amino acid residue 65 of SEQ ID NO:2; c) a polypeptide having the sequence of amino acid residue 21 to amino acid residue 65 of SEQ ID NO:2; d) a polypeptide having the sequence of amino acid residue 1 to amino acid residue 67 of SEQ ID NO:10; e) a polypeptide having the sequence of amino acid residue 21 to amino acid residue 67 of SEQ ID NO:10; and f) a polypeptide having the sequence of amino acid residue 23 to amino acid residue 67 of SEQ ID NO:10; wherein the polypeptide has cysteine residues corresponding to amino acid residues 33, 40, 45, 55, 62 and 63 of SEQ ID NOs:2 or 10.
Within another aspect the invention provides an isolated polynucleotide molecule encoding a protein having cysteine residues corresponding to amino acid residues 33, 40, 45, 55, 62 and 63 of SEQ ID NO:10, the polynucleotide molecule consisting of a coding strand and a complementary non-coding strand, wherein the polynucleotide comprises a nucleotide sequence that is at least 80% identical to the sequence of a polynucleotide selected from the group consisting of: a) a polynucleotide as shown in SEQ ID NO:9 from nucleotide 220 to nucleotide 420; b) a polynucleotide as shown in SEQ ID NO:9 from nucleotide 280 to nucleotide 420; and c) a polynucleotide as shown in SEQ ID NO:9 from nucleotide 286 to nucleotide 420.
Within yet another aspect is provided an isolated polynucleotide molecule encoding a protein having cysteine residues corresponding to amino acid residues 33, 40, 45, 55, 62 and 63 of SEQ ID NO:10, the polynucleotide molecule consisting of a coding strand and a complementary non-coding strand, wherein the polynucleotide comprises a nucleotide sequence as shown in SEQ ID NO:11.
Within another aspect is provided an isolated polynucleotide molecule encoding a polypeptide selected from the group consisting of: a) amino acid residue 30 to amino acid residue 63 of SEQ ID NO:2; b) amino acid residue 31 to amino acid residue 63 of SEQ ID NO:2; c) amino acid residue 30 to amino acid residue 64 of SEQ ID NO:2; d) amino acid residue 31 to amino acid residue 64 of SEQ ID NO:2; and e) a polypeptide chosen from SEQ ID NOs:14-72.
The invention also provides an isolated polynucleotide molecule selected from the group consisting of:
a) nucleotide 88 to nucleotide 189 of SEQ ID NO:1;
b) nucleotide 88 to nucleotide 192 of SEQ ID NO:1;
c) nucleotide 91 to nucleotide 189 of SEQ ID NO:1;
d) nucleotide 91 to nucleotide 192 of SEQ ID NO:1;
e) nucleotide 88 to nucleotide 189 of SEQ ID NO:4;
f) nucleotide 88 to nucleotide 192 of SEQ ID NO:4;
g) nucleotide 91 to nucleotide 189 of SEQ ID NO:4 and
h) nucleotide 91 to nucleotide 192 of SEQ ID NO:4.
Within still another aspect is provided an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide selected from the group consisting of: a) a protein of claim 1; a) amino acid residue 30 to amino acid residue 63 of SEQ ID NO:2; b) amino acid residue 31 to amino acid residue 63 of SEQ ID NO:2; c) amino acid residue 30 to amino acid residue 64 of SEQ ID NO:2; d) amino acid residue 31 to amino acid residue 64 of SEQ ID NO:2; and e) a polypeptide chosen from SEQ ID NOs:14-72; and a transcription terminator. Within one embodiment the DNA segment further encodes a secretory signal sequence operably linked to the protein. Within a related embodiment the secretory signal sequence is selected from the group consisting of: a) a polypeptide having the sequence of amino acid residue 1 to amino acid residue 18 of SEQ ID NO:2; b) a polypeptide having the sequence of amino acid residue 1 to amino acid residue 20 of SEQ ID NO:2; c) a polypeptide having the sequence of amino acid residue 1 to amino acid residue 20 of SEQ ID NO:10; and d) a polypeptide having the sequence of amino acid residue 1 to amino acid residue 22 of SEQ ID NO:10.
Within another aspect the invention provides a cultured cell into which has been introduced an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as described above; and a transcription terminator; wherein the cell expresses the protein encoded by the DNA segment.
Within a further aspect is provided a method of producing a protein comprising: culturing a cell into which has been introduced an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as described above; and a transcription terminator; whereby the cell expresses the protein encoded by the DNA segment; and recovering the expressed protein.
The invention also provides an oligonucleotide probe or primer comprising at least 14 contiguous nucleotides of a polynucleotide of SEQ ID NO:1 or a sequence complementary to SEQ ID NO:11.
The invention further provides a method of treating a microbial-related disease comprising administering to a mammal a therapeutically effective amount of a polypeptide selected from the group consisting of: a) a polypeptide of SEQ ID NO:2; b) a polypeptide of SEQ ID NO:10; c) a polypeptide chosen from SEQ ID NOs:14-72; h) amino acid residue 30 to amino acid residue 63 of SEQ ID NO:2; i) amino acid residue 31 to amino acid residue 63 of SEQ ID NO:2; j) amino acid residue 30 to amino acid residue 64 of SEQ ID NO:2; k) amino acid residue 31 to amino acid residue 64 of SEQ ID NO:2l) amino acid residue 20 to amino acid residue 67 of SEQ ID NO:10 and m) amino acid residue 22 to amino acid residue 67 of SEQ ID NO:10; whereby said polypeptide ameliorates said disease. Within one embodiment the microbial-related disease is associated with the eye. Within a related embodiment the microbial-related disease is conjunctivitis. Within another embodiment the microbial-related disease is associated with the ear.
Also provided is a method of contraception comprising administering to a mammal a therapeutically effective amount of a polypeptide selected from the group consisting of: a) a polypeptide of SEQ ID NO:2; b) a polypeptide of SEQ ID NO:10; c) a polypeptide chosen from SEQ ID NOs:14-72; h) amino acid residue 30 to amino acid residue 63 of SEQ ID NO:2; i) amino acid residue 31 to amino acid residue 63 of SEQ ID NO:2; j) amino acid residue 30 to amino acid residue 64 of SEQ ID NO:2; k) amino acid residue 31 to amino acid residue 64 of SEQ ID NO:2; l) amino acid residue 20 to amino acid residue 67 of SEQ ID NO:10 and m) amino acid residue 22 to amino acid residue 67 of SEQ ID NO:10.